Activated formylglycine-generating enzymes and methods of producing and using the same

Patent metadata

Field	Value
Publication number	US-9951367-B2
Application number	US-201514975403-A
Country	US
Kind code	B2
Filing date	Dec 18, 2015
Priority date	Feb 5, 2015
Publication date	Apr 24, 2018
Grant date	Apr 24, 2018

Abstract

The present disclosure provides activated formylglycine-generating enzymes (FGE), methods of producing activated FGE, and their use in methods of producing a protein comprising a formylglycine (FGly) residue. The methods of producing activated FGE, as well as methods of use of activated FGE in producing FGly-containing proteins, include both cell-based and cell-free methods. Compositions and kits that find use, e.g., in practicing the methods of the present disclosure are also provided.

First claim

What is claimed is:

1. A method of producing a protein comprising a formylglycine residue, the method comprising:
expressing a combining an activated formylglycine-generating enzyme (FGE) and a protein comprising an FGE recognition site in a cell-free reaction mixture comprising Cu 2+ to activate the FGE, under conditions in which the activated FGE converts a cysteine residue or a serine residue of the FGE recognition site to a formylglycine residue, to produce a protein comprising a formylglycine residue.

2. The method according to claim 1 , wherein the activated FGE and the protein comprising the FGE recognition site are combined in a reaction mixture comprising a reducing agent.

3. The method according to claim 2 , wherein the reducing agent promotes conversion of the cysteine residue or the serine residue of the FGE recognition site to the formylglycine residue.

4. The method according to claim 3 , wherein the reducing agent is 2-mercaptoethanol.

5. The method according to claim 1 , wherein the Cu 2+ is provided by a source of Cu 2+ selected from copper sulfate, copper citrate, copper tartrate, Fehling's reagent, and Benedict's reagent.

6. The method according to claim 1 , wherein the source of Cu 2+ is copper sulfate.

7. The method according to claim 1 , wherein the FGE is an N-terminally truncated FGE.

8. The method according to claim 7 , wherein the FGE is a N-terminally truncated human FGE.

9. The method according to claim 1 , wherein the protein is an antibody, an antibody fragment, a ligand, an enzyme, or an antigen.

10. The method according to claim 1 , wherein the protein is an antibody or antibody fragment.

11. The method according to claim 10 , wherein the antibody or antibody fragment is selected from the group consisting of: an IgG or fragment thereof, a Fab, a F(ab′)2, a Fab′, an Fv, an ScFv, a bispecific antibody or fragment thereof, a diabody or fragment thereof, a chimeric antibody or fragment thereof, a monoclonal antibody or fragment thereof, a humanized antibody or fragment thereof, and a fully human antibody or fragment thereof.

12. The method according to claim 10 , wherein the antibody specifically binds to a tumor-associated antigen or a tumor-specific antigen.

13. The method according to claim 12 , wherein the tumor associated antigen or tumor-specific antigen is selected from the group consisting of: HER2, CD19, CD22, CD30, CD33, CD56, CD66/CEACAM5, CD70, CD74, CD79b, CD138, Nectin-4, Mesothelin, Transmembrane glycoprotein NMB (GPNMB), Prostate-Specific Membrane Antigen (PSMA), SLC44A4, CA6, and CA-IX.

14. The method according to claim 1 , wherein the protein is a ligand.

15. The method according to claim 14 , wherein the ligand is a growth factor.

16. The method according to claim 14 , wherein the ligand is a hormone.

Assignees

• Scherer Technologies Llc R P

Inventors

• Rabuka David
• Dehart Gregory W
• Holder Patrick
• Baker Jeanne

Classifications

• A61P31/04

  Antibacterial agents · CPC title

• A61P31/10

  Antimycotics · CPC title

• A61P35/00

  Antineoplastic agents · CPC title

• A61P31/12

  Antivirals · CPC title

• C12N9/0051

  acting on a sulfur group of donors (1.8) · CPC title